Viola uliginosa Besser is a European violet having its main distribution range in the Baltic Sea region. Today it is considered endangered and threatened. Species of Violaceae from different genera and sections are known to produce cyclotides, cyclic polypeptides of much interest due to their medicinal properties and chemical structure. The present study introduced a rare species of violet (V. uliginosa) to in vitro culture for biodiversity protection and as a model for cyclotide biosynthesis research in the Violaceae. Leaf and petiole fragments were cultured on MS medium solidified with agar and supplemented with different concentrations of plant growth regulators: TDZ, KIN and 2,4-D. Direct and indirect (via callus) organogenesis was induced on MS supplemented with TDZ (0.5 or 1 mg l−1) or with equal concentrations (2 mg l−1) of KIN and 2,4-D, followed by callus transfer on 1 mg l−1 TDZ. Shoots were rooted on MS with 2 % sucrose and 0.5 mg l−1 IBA and acclimatized. AFLP marker polymorphism was low but flow cytometry revealed that a large share of the obtained regenerants were tetraploid (2C = 4x = 2.7–2.8 pg), unlike the maternal diploid plants (2C = 2x = 1.4 pg). Eleven different cyclotides were distinguished in the aerial parts of maternal plants. Cyclotide production was significantly higher in tetraploid than in diploid plants regenerated in vitro.